Exposure to many chemotherapeutic agents results in DNA damage that frequently occurs at the N7 position of guanine (N7-Gua). Although these adducts can serve as biomarkers for these treatments, as well as exposures to environmental carcinogens, there are no data supporting a role for N7-Gua adducts in promoting mutagenesis. However, there are compelling data that implicate the ring-opened form of alkylated N7-Gua adducts, N[5]-alkyl-2,6-diamino-4-hydroxyformamido-pyrimidine (alkyl-Fapy) in mutagenesis and carcinogenesis. The goal of the Program Project is to determine the structural, molecular, and cellular bases for mutagenesis caused by a variety of alkyl-Fapy-Gua adducts that are produced during therapeutic treatments with temozolomide, thioTEPA and nitrogen mustard. The goals of Project 2 are to investigate the mutagenic potential of these adducts, identify the DNA polymerase(s) that generate errors as a consequence of lesion bypass, and invesfigate the repair systems that modulate mutation frequencies and spectra. Specific Aim 1 will establish the mutagenic potential of these Fapy-dG DNA adducts by evaluating the frequency and spectra of mutations formed following replication in wild-type primate cells. It is hypothesized that both the mutation frequency and spectra will be significantly modulated by the local sequence context of the adduct, the steady-state stereochemical equilibrium ofthe adduct at the replication fork, and the size of the N[5]-alkyl-Fapy-dG adduct. The roles of various DNA mismatch repair mechanisms in limifing mutagenesis will also be assessed. The objective of Specific Aim 2 is to identify the DNA polymerases that are responsible for the low and high fidelity bypass of the N[5]-alkyl-Fapy adducts. It is hypothesized that pol K and possibly pol n are primarily responsible for TLS of these adducts and this will be tested using a multidisciplinary approach of shRNA-mediated depletion of individual polymerases and specific small molecule inhibitors. The objective of Specific Aim 3 is to determine the DNA repair mechanism(s) that modulate the mutagenic potential of N[5]-alkyl-Fapy-dG adducts. It is hypothesized that base and nucleotide excision repair will modulate mutagenesis in cells that are shRNA-depleted for repair proteins. Specific aim 4 will investigate repair and replication of DNAs containing interstrand DNA cross-links that arise from nitrogen mustards. It is hypothesized that interactions between DNA repaired translesion synthesis polymerases can leed to cellular resistance to these agents.